Kurjan et al. (1982) Cell 30:933-943 discloses the first cloning and sequencing of a gene encoding a yeast .alpha.-factor precursor gene. Kurjan et al., U.S. Pat. No. 4,546,082, also reports the cloning of this gene, and suggests that the .alpha.-factor leader sequence can be employed to direct the secretion of heterologous proteins in yeast. The patent, however, does not contain data which would indicate that the patentees ever successfully employed the .alpha.-factor leader to express and secrete a heterologous protein in yeast.
EPO Publication No. 116,201 discloses the first successful application of the .alpha.-factor leader to direct the expression and secretion of an heterologous protein, epidermal growth factor, from a transformed yeast. Subsequent to this work, there have been additional reports of the expression of heterologous proteins in yeast employing the .alpha.-factor leader. See, e.g., Elliott et al. (1983) Proc. Nat'l Acad. Sci. USA 80:7080-7084; Bitter et al. (1984) Proc. Nat'l Acad. Sci. USA 81:5330-5334; Smith et al. (1985) Science 229:1219-1229; EPO Publication Nos. 114,695; 123,228; 123,294; 123,544; 128,773; and 206,783. See also Brake et al. in Protein Transport and Secretion, p.103 (J. M. Gething ed. 1984).
The expression systems in the above reports produce a full-length .alpha.-factor leader fused to a heterologous protein. While the above work demonstrates that the .alpha.-factor expression system is widely useful, it is not generally predictable prior to performing the experiment whether a particular heterologous protein will be successfully secreted, processed and biologically active. See, e.g., EPO 206,783, supra, pp. 2-5; Rothblatt et al. (1987) EMBO J. 6:3455-3463; V. L. MacKay, "Secretion of Heterologous Proteins in Yeast" (in press).
There have been several reports, based on unpublished data, that deletions from the "pro" region of the .alpha.-factor leader (between the signal peptide and the first spacer) causes substantial declines in the amount of non-yeast protein secreted by yeast transformed the heterologous constructs. Sidhu et al. (1987) Gene 54:175-184, reports that yeast acid phosphatase (PHO5) is secreted into the medium from a heterologous construct employing both a full-length .alpha.-factor leader, and a truncated .alpha.-factor leader, but that expression levels are 3-4.times. less than for the PHO5 gene under the control of its homologous leader. It has also been reported that deletions in the prepro-.alpha.-factor precursor gene results in substantial declines in secretion of the native .alpha.-factor peptide. See, e.g., V. L. MacKay, supra; Rothblatt et al., supra.
A need exists, therefore, to improve the .alpha.-factor expression system, particularly for applications to non-yeast proteins that are not efficiently produced by current .alpha.-factor expression constructs.